Eva E. Prieschl

Sphingolipids: second messengers, mediators and raft constituents in signaling

Recently, evidence has accumulated to show that sphingolipids exert an important function in signaling. These lipids serve as intracellular second messengers and as extracellular mediators. Furthermore, glycosylated sphingolipids are essential components of membrane rafts, which serve as platforms for the initiation of signaling cascades. Here, Eva Prieschl and Thomas Baumruker summarize current findings in leukocytes illustrating these different facets.

Characterization of CDw92 as a Member of the Choline Transporter-Like Protein Family Regulated Specifically on Dendritic Cells

CDw92 is a 70-kDa surface protein broadly expressed on leukocytes and endothelial cells. In this manuscript, we present the molecular cloning of the CDw92 molecule by using a highly efficient retroviral expression cloning system. Sequence analysis of the CDw92 cDNA revealed a length of 2679 bp. The 1959-bp open reading frame encodes a protein of 652 amino acids. Computational analysis of the CDw92 protein sequence indicates 10 transmembrane domains, three potential N-linked glycosylation sites, and an amino acid stretch in the C-terminal region that is related to the immunoreceptor tyrosine-based inhibitory motif. Comparison of the sequence of the CDw92 clone presented in this study with various database entries show that it is a C-terminal variant of human choline transporter-like protein 1, a member of a recently identified family of multitransmembrane surface proteins. Furthermore, we found that CDw92 is stably expressed on monocytes, PBLs, and endothelial cells, as we did not yet find modulation of expression by various stimuli on these cells. In contrast to this factor-independent expression of CDw92, we detected a specific regulation of CDw92 on monocyte-derived dendritic cells (Mo-DCs). Maturation of Mo-DCs by ionomycin or calcium ionophore resulted in down-regulation of CDw92 and incubation of these cells with IL-10 in a specific re-expression. Moreover, targeting of CDw92 on LPS-treated Mo-DCs by CDw92 mAb VIM15b augmented the LPS-induced IL-10 production 2.8-fold. Together, these data suggest a crucial role of the CDw92 protein in the biology and regulation of the function of leukocytes in particular DCs.

Suppression of early T-cell-receptor-triggered cellular activation by the Janus kinase 3 inhibitor WHI-P-154.

Background. Therapeutic targeting of Janus kinase 3 (JAK3) has received particular attention, because it is associated with the common &ggr; signaling of cytokine receptors and thus vitally influences T-cell growth and survival. Recent evidence, however, indicates a critical role for JAK3 in signaling linked to the T-cell antigen receptor. Methods. In this study we investigated whether targeting JAK3 with a rationally designed inhibitor affects early T-cell activation events. T cells were stimulated by CD3 and CD28 cross-linking, and interleukin (IL)-2 production, activation marker expression, increase of free intracellular Ca2+ concentration, activation of the extracellular-related kinase, and nuclear translocation of transcription factors were evaluated. Results. We found that JAK3 inhibitor treatment dramatically impaired T-cell–receptor (TCR)-induced IL-2 production, surface activation marker expression (CD69, CD154), and homotypic T-cell aggregation. Accordingly, mRNA production of IL-2, interferon-&ggr;, and IL-10 was profoundly inhibited. Molecular analysis revealed that TCR-triggered phosphorylation of phospholipase C-&ggr;1, increase in cytoplasmic Ca2+ concentration, and activation of extracellular-related kinase were markedly reduced by the JAK3 inhibitor, resulting in substantially decreased DNA binding of nuclear factor of activated T cells and alkaline phosphatase-1 and subsequent IL-2 promoter activation. Remarkably, on TCR-independent stimulation, IL-2 production, CD69 expression, and blast formation were completely insensitive to JAK3 inhibitor treatment. Conclusion. These data indicate that pharmacologic targeting of JAK3 uncouples early TCR-triggered signaling from essential downstream events, which may have important implications for the use of such compounds in T-cell–mediated disorders such as allograft rejection or graft-versus-host disease.

Effects of phosphatidylinositol-3-kinase inhibitors on degranulation and gene induction in allergically triggered mouse mast cells

BACKGROUND Phosphatidylinositol-3-kinase (PI3-kinase) comprises an essential component in a number of signaling cascades, primarily of the growth factor type. Two specific inhibitors, wortmannin and demethoxyviridin (DMV), are widely used to block signaling via this molecule and link certain receptors to the PI3-kinase pathway. METHODS We have studied the extent of involvement of PI3-kinase in signaling events by Fc epsilonRI in mast cells using a mouse mast cell line as a model system. This was done using beta-hexosaminidase release assays, a leukotriene ELISA, transient transfections with reporter gene constructs of TNF alpha and MARC, and in addition a TNF alpha ELISA. RESULTS Consistent with previously published data in the rat basophilic cell line RBL-2H3, we find that wortmannin as well as DMV prevent the degranulation reaction in the mouse mast cell line CPII. DMV also inhibits the release of leukotrienes, leading to the conclusion that Fc epsilonRI activates PI3-kinase which then mediates these reactions. On the contrary, however, lymphokine and chemokine induction at the gene and protein level is not inhibited, suggesting that the activation of this gene set in mast cells is independent of PI3-kinase. CONCLUSION PI3-kinase is activated in our mast cell model system via cross-linking of the Fc epsilonRI. This reaction is clearly necessary for the degranulation process and the release of leukotrienes. Activation of lymphokine and chemokine genes as well as secretion of their gene products are not triggered along the PI3-kinase signaling pathway. This is in agreement with our previous findings, showing that the MAP kinase pathway and Ca2+ influx are both involved in gene activation in this cell type.

The Role of Sphingosine Kinase in the Signaling Initiated at the High-Affinity Receptor for IgE (FcεRI) in Mast Cells

Over the last few years, sphingolipids have emerged as an additional class of lipids participating in signaling events in various cell types. The best-investigated examples so far are ceramide and sphingosine-1-phosphate. Ceramide-activated protein kinase and sphingosine kinase are two enzymes which respond to and generate these mediators. In particular, sphingosine kinase, its substrate sphingosine and the product sphingosine-1-phosphate have recently been implicated in the signaling cascades initiated at the FcεRI of mast cells. High intracellular levels of sphingosine seem to serve as an ‘intracellular’ inhibitor which is ‘deactivated’ by the action of sphingosine kinase, due to the conversion to sphingosine-1-phosphate. One mode of action of the inhibitory process in this cell type is prevention of the activation of the mitogen-activated protein (MAP) kinase pathway. Sphingosine-1-phosphate itself, the product of this enzymatic reaction, is believed to lead to Ca2+ mobilization and to stimulate the MAP kinase pathway. The existence and function of this second messenger explains the ‘IP3 gap’ described in mast cells after FcεRI activation. Therefore, a picture emerges whereby the balance of these two lipid molecules seems to be decisive for the activation of mast cells by IgE plus antigen, with sphingosine kinase acting as a permissive switch for stimulation.

TNF–· and IL–5 Gene Induction in IgE plus Antigen–Stimulated Mast Cells Require Common and Distinct Signaling Pathways

Background: Mast cells produce a variety of cytokines and chemokines in a timely and tightly controlled fashion if stimulated via the FcεRI. Evidence is accumulating that the transcriptional induction of the corresponding genes and the release of these mediators are dependent on common and mediator–specific components of the signal transduction and transcription factor machinery. Methods: We addressed this issue by comparing the effects of mitogen activated protein (MAP) kinase pathway inhibitors and protein kinase C (PKC) inhibitors on the induction of TNF–α and IL–5 after IgE plus antigen (Ag) stimulation in CPII mouse mast cells using Western blot analyses and transient transfections of reporter gene plasmids. Results: TNF–α shows a strict dependence on the MAP kinase pathway, while IL–5 is either activated by PMA–dependent PKCs or along the MAP kinase pathway. In addition, both mediators are sensitive to PKCμ inhibition, suggesting involvement of this atypical, non–PMA dependent PKC in the overall induction process. Conclusion: While the two cytokines were recently shown to be regulated by a member of the nuclear factor of activated T–cells (NF–AT) transcription factor family, activator protein 1 (AP1) was identified as a cofactor at the TNF–α promoter while a GATA family member comprised the cofactor at the IL–5 promoter. This suggests that the differences in requirement for signal transduction cascades are the result of a different usage of NF–AT cofactors for transcription of each cytokine in mast cells.

Glycosphingolipid-Induced Relocation of Lyn and Syk into Detergent-Resistant Membranes Results in Mast Cell Activation

Sphingosine, sphingosine-1-phosphate, and the more complex sphingolipid ceramide exert strong immunomodulatory effects on a variety of leukocytes. However, little is known regarding such a potential of glycosphingolipids, a class of sugar derivatives of sphingosine. Here we demonstrate that galactosylsphingosine, one of the smallest representatives of this group, accumulates in the detergent-resistant membranes resulting in the relocation of the tyrosine kinases Lyn and Syk into this compartment. The result of this is an enhanced tyrosine phosphorylation and kinase activity leading to priming and activation of mast cells by conveying a weak yet significant activation of the mitogen-activated protein kinase pathway(s). In comparison to IgE/Ag triggering, galactosylsphingosine stimulates the mitogen-activated protein kinase pathway more rapidly and favors c-Jun NH2-terminal kinase 1 activation over extracellular signal-regulatory kinase 1 and 2. At the transcription factor level, this “ultratransient signaling event” results in an activation of JunD as the predominant AP-1 component. In this respect, the effects of galactosylsphingosine are clearly distinct from the signaling elicited by other sphingolipids without the sugar moiety, such as sphingosine-1-phosphate.

Major histocompatibility complex class II– fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responses

Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I‐restricted fashion, do not exhibit anti‐MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC‐like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC‐specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti‐T‐cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti‐CD3‐induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of FcγR on 80/1 DC and that the requirement for co‐stimulation depends on the intensity of T‐cell receptor signalling.

Mast cell signalling: a patent review

Mast cells, basophils and eosinophils are the effector cells in Type I (immediate hypersensitivity or immunoglobulin E (IgE)-associated) allergic disorders. The release of granule-associated/lipid mediators as well as the production of various cytokines/chemokines is intimately linked to the acute and late phase symptoms observed in atopic patients. Perhaps standing at the beginning of many if not all chronic allergic inflammatory conditions with high specific IgE levels. Binding of IgE to the high affinity FceRI receptor, which is constitutively expressed on mast cells, basophils, and some eosinophils, and its crosslinking via allergen initiates the resulting pathology. The relative specificity of the ligand (IgE)/receptor (FceRI) interaction and the discovery of specific intracellular signalling cascades in other cell types has prompted efforts to interfere at these levels with the activation of those effector cells. Here we report on the pharmaceutical endeavours over the last 5 years - reflected in th...

FcεRI-Mediated Activation of NF-AT

The term nuclear factor of activated T-cells (NF-AT) appeared in the scientific literature in 1988 to describe a protein visualized in gel shift assays binding to the ARRE (antigen receptor response element) of the IL-2 promoter and forming a complex called NF-IL2-E.1 In contrast to many other binding activities and transcription factors that were first described in one specific cell type and later found to be of broader significance, NF-AT has remained as a more or less T-cell-induction-specific DNA-binding activity.1–5 It escaped a detailed molecular characterization because all efforts to clone the corresponding gene or cDNA failed.